There is a growing demand for binding molecules consisting of amino acids which are not immunoglobulins. While until now antibodies represent the best-established class of binding molecules there is still a need for new binding molecules in order to target ligands with high affinity and specificity since immunoglobulin molecules suffer from major drawbacks. Although they can be produced quite easily and may be directed to almost any target, they have a quite complex molecular structure. There is an ongoing need to substitute antibodies by smaller molecules which can be handled in an easy way. These alternative binding agents can be beneficially used for instance in the medical fields of diagnosis, prophylaxis and treatment of diseases.
Proteins having relatively defined 3-dimensional structures, commonly referred to as protein scaffolds, may be used as starting material for the design of said alternative binding agents. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomisation is often carried out to produce a library of proteins from which the specific binding molecules may be selected. Molecules with a smaller size than antibodies and a comparable or even better affinity towards a target antigen are expected to be superior to antibodies in terms of pharmacokinetic properties and immunogenicity.
A number of previous approaches do use protein scaffolds as starting material of binding proteins. For example, in WO 99/16873 modified proteins of the lipocalin family (so-called Anticalins) exhibiting binding activity for certain ligands were developed. The structure of peptides of the lipocalin family is modified by amino acid replacements in their natural ligand binding pocket using genetic engineering methods. Like immunoglobulins, the Anticalins can be used to identify or bind molecular structures. In a manner analogously to antibodies, flexible loop structures are modified; these modifications enable the recognition of ligands different from the natural ones.
WO 01/04144 describes the artificial generation of a binding domain on the protein surface in beta sheet structural proteins per se lacking a binding site. By means of this de novo generated artificial binding domain e.g. variations in γ-crystallin—an eye lens structural protein—can be obtained which interact with ligands with high affinity and specificity. In contrast to the modification of binding sites which are already present and formed from flexible loop structures as mentioned above for Anticalins, these binding domains are generated de novo on the surface of beta sheets. However, WO 01/04144 only describes the alteration of relatively large proteins for the generation of novel binding properties. Due to their size the proteins according to WO 01/04144 can be modified on the genetic engineering level only by methods which require some effort. Furthermore, in the proteins disclosed so far only a relatively small proportion by percentage of the total amino acids was modified in order to maintain the overall structure of the protein. Therefore, only a relatively small region of the protein surface is available which can be utilized for the generation of binding properties that did not exist previously. Moreover, WO 01/04144 discloses only the generation of a binding property to γ-crystallin.
WO 04/106368 describes the generation of artificial binding proteins on the basis of ubiquitin proteins. Ubiquitin is a small, monomeric, and cytosolic protein which is highly conserved in sequence and is present in all known eukaryotic cells from protozoans to vertebrates. In the organism, it plays a crucial role in the regulation of the controlled degradation of cellular proteins. For this purpose, the proteins destined for degradation are covalently linked to ubiquitin or polyubiquitin chains during their passage through a cascade of enzymes and are selectively degraded because of this label. According to recent results, ubiquitin or the labelling of proteins by ubiquitin, respectively, plays an important role also in other cellular processes such as the import of several proteins or the gene regulation thereof.
Besides the clarification of its physiological function, ubiquitin is a research object primarily because of its structural and protein-chemical properties. The polypeptide chain of ubiquitin consists of 76 amino acids folded in an extraordinarily compact α/β structure (Vijay-Kumar, 1987): almost 87% of the polypeptide chain is involved in the formation of the secondary structural elements by means of hydrogen bonds. Secondary structures are three and a half alpha-helical turns as well as an antiparallel β sheet consisting of four strands. The characteristic arrangement of these elements—an antiparallel β sheet exposed of the protein surface onto the back side of which an alpha helix is packed which lies vertically on top of it—is generally considered as so-called ubiquitin-like folding motif. A further structural feature is a marked hydrophobic region in the protein interior between the alpha helix and the β sheet.
Because of its small size, artificial preparation of ubiquitin can be carried out both by chemical synthesis and by means of biotechnological methods. Due to the favourable folding properties, ubiquitin can be produced by genetic engineering using microorganisms such as Escherichia coli in relatively large amounts either in the cytosol or in the periplasmic space. Because of the oxidizing conditions predominating in the periplasm the latter strategy generally is reserved for the production of secretory proteins. Due to the simple and efficient bacterial preparation ubiquitin can be used as a fusion partner for other foreign proteins to be prepared for which the production is problematic. By means of fusion to ubiquitin an improved solubility and thereby an improved production yield can be achieved.
Compared to antibodies or other alternative scaffolds, artificial binding proteins on the basis of ubiquitin proteins (also referred to as AFFILIN®) have many advantages: small size, high stability, high affinity, high specificity, cost effective microbial manufacturing, and adjustment of serum half life. However, there is still a need to further develop those proteins in terms of new therapeutic approaches with high affinities to specific targets. While WO 05/05730 generally describes the use of ubiquitin scaffolds in order to obtain artificial binding proteins, no solution is provided on how to modify an ubiquitin protein in order to obtain a specific and high affinity binding to the ED-B of fibronectin.
WO 2008/022759 describes recombinant binding proteins wherein the Src homology 3 domain (SH3) of the FYN kinase is used for obtaining new binding proteins. It was found that the target specificity can be designed by mutating the RT loop and/or the Src loop in order to develop protein therapeutics and/or diagnostics. Like in lipocalins used as scaffold, the amino acid residues to be mutagenized lie within the variable and flexible loop regions mimicking the principle underlying the antibody/antigen binding function. This overall flexibility of the interaction site by which antibodies bind the epitope is a mainly enthalpically driven process; this process, however, leads to an unfavorable entropic contribution by loss of mobility upon association of the flexible complementarity determining region. Contrary thereto, using ubiquitin as a scaffold, the present inventors did not change amino acid residues primarily within the flexible loop regions but within the rigid and inflexible β strands of a β sheet region or closely adjacent to the beta strands. The advantage of selecting amino acid residues within the inflexible and rigid β strands or closely adjacent to the beta strands of ubiquitin as binding regions for ED-B is inter alia the following: The binding partners are thought to already present a complementary geometry appropriate for tight binding. Consequently, these interactions involve complementarity in shape, charge and hydrophilic/hydrophobic elements of the more rigid structures of the binding partners. These rigid body interactions optimize the interface and accommodate biological function.
Fibronectins (FN) are an important class of high molecular weight extracellular matrix glycoproteins abundantly expressed in healthy tissues and body fluids. Their main role consists in facilitating the adhesion of cells to a number of different extracellular matrices. The presence of fibronectins on the surface of non-transformed cells in culture as well as their absence in the case of transformed cells resulted in the identification of fibronectins as important adhesion proteins. They interact with numerous various other molecules, e.g. collagen, heparan sulphate-proteoglycans and fibrin and thus regulate the cell shape and the creation of the cytoskeleton. In addition, they are responsible for cell migration and cell differentiation during embryogenesis. They also play an important role in wound healing, in which they facilitate the migration of macrophages and other immune cells and in the formation of blood clots by enabling the adhesion of blood platelets to damaged regions of the blood vessels.
The extra-domain B (ED-B) of fibronectin is a small domain which is inserted by alternative splicing of the primary RNA transcript into the fibronectin molecule. The molecule is either present or omitted in fibronectin molecules of the extracellular matrix and represents a one of the most selective markers associated with angiogenesis and tissue remodelling, as it is abundantly expressed around new blood vessels, but undetectable in virtually all normal adult tissues (except for uterus and ovaries). ED-B is known to be involved primarily in cancer. High levels of ED-B expression were detected in primary lesions as well as metastatic sites of many human solid cancer entities, including breast, non-small cell lung, colorectal, pancreatic, human skin, hepatocellular, intracraneal meningeoma, glioblastoma (Menrad u. Menssen, 2005). Furthermore, ED-B can be bound to diagnostic agents and be favorably used as diagnostic tool. One example is its use in molecular imaging of e.g. atherosclerotic plaques and detection of cancer, e.g. by immunoscintigraphy of cancer patients. Plenty of further diagnostic uses are conceivable.
The amino acid sequence of 91 amino acids of human extra-domain B (ED-B) of fibronectin is shown in SEQ ID NO: 2. For expression of the protein, a start methionin has to be added. ED-B is abundant in mammals, e.g. in rodents, cattle, primates, carnivore, human etc. Examples of animals in which there is a 100% sequence identity to human ED-B are Rattus norvegicus, Bos taurus, Mus musculus, Equus caballus, Macaca mulatta, Canis lupus familiaris, and Pan troglodytes. 
ED-B specifically accumulates in neo-vascular structures and represents a target for molecular intervention in cancer. A number of antibodies or antibody fragments to the ED-B domain of fibronectin are known in the art as potential therapeutics for cancer and other indications (see, for example, WO 97/45544, WO 07/054,120, WO 99/58570, WO 01/62800). Human single chain Fv antibody fragment ScFvL19 (also referred to as L19) is specific to the ED-B domain of fibronectin and has been verified to selectively target tumor neovasculature, both in experimental tumor models and in patients with cancer. Furthermore, conjugates comprising an anti-ED-B antibody or an anti-ED-B antibody fragment with cytokines such as IL-12, IL-2, IL-10, IL-15, IL-24, or GM-CSF have been described for targeting drugs for the manufacture of a medicament for inhibiting particularly cancer, angiogenesis, or neoplastic growth (see, for example, WO06/119897, WO07/128,563, WO01/62298). The selective targeting of neovasculature of solid tumors with anti-ED-B antibodies or anti-ED-B antibody fragments such as L19 conjugated to an appropriate effector function such as a cytotoxic or an immunostimulating agent has proven to be successful in animal experiments. For the therapy of pancreatic cancer, fusion proteins comprising an Interleukin-2 part (IL-2) and an anti-ED-B antibody part were combined with the small molecule Gemcitabine (2′-deoxy-2′,2′-difluorocytidine) (see, for example, WO 07/115,837).
The above-discussed prior art documents describe the use of various protein scaffolds including antibodies to generate new ED-B binding proteins. Targeting ED-B with currently available compounds has certain disadvantages. Smaller molecules (such as hetero-multimeric ubiquitin-based ED-B binding proteins of this invention) with a comparable or even higher affinity towards the ED-B antigen are expected to have significant advantages to antibodies or other binding proteins.
Since cancer represents one of the leading causes for death worldwide, there is a growing need for improved agents for treating cancer. Current chemotherapeutic agents and radiation treatment suffer from poor selectivity and most chemotherapeutic agents do not accumulate at the tumor site and thus fail to achieve adequate levels within the tumor. There is a strong medical need to effectively treat cancer.
It is thus an object of the present invention to provide hetero-multimeric binding proteins based on ubiquitin being able to bind specifically with very high affinity to the extracellular domain of fibronectin (ED-B). It is a further object of the present invention to identify and provide novel binding proteins with very high binding specificity to ED-B for example for use in the treatment of cancer. Furthermore, a method shall be provided in order to produce said hetero-multimeric binding molecules.
The above-described objects are solved by the subject-matter of the enclosed independent claims. Preferred embodiments of the invention are included in the dependent claims as well as in the following description, examples and figures.